RESUMO

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site's conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus' mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

RESUMO

AIMS: In December 2019, the Coronavirus disease-2019 (COVID-19) virus has emerged in Wuhan, China. In this research, the first resolved COVID-19 crystal structure (main protease) was targeted in a virtual screening study by of FDA approved drugs dataset. In addition, a knowledge gap in relations of COVID-19 with the previously known fatal Coronaviruses (CoVs) epidemics, SARS and MERS CoVs, was covered by investigation of sequence statistics and phylogenetics. MATERIALS AND METHODS: Molecular modeling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 main protease were investigated. KEY FINDINGS: COVID-19 Mpro formed a phylogenetic group with SARS CoV that was distant from MERS CoV. The identity% was 96.061 and 51.61 for COVID-19/SARS and COVID-19/MERS CoV sequence comparisons, respectively. The top 20 drugs in the virtual screening studies comprised a broad-spectrum antiviral (ribavirin), anti-hepatitis B virus (telbivudine), two vitamins (vitamin B12 and nicotinamide) and other miscellaneous systemically acting drugs. Of special interest, ribavirin had been used in treating cases of SARS CoV. SIGNIFICANCE: The present study provided a comprehensive targeting of the first resolved COVID+19 structure of Mpro and found a suitable save drugs for repurposing against the viral Mpro. Ribavirin, telbivudine, vitamin B12 and nicotinamide can be combined and used for COVID treatment. This initiative relocates already marketed and approved safe drugs for potential use in COVID-treatment.

RESUMO

We prepared the three-dimensional model of the SARS-CoV-2 (aka 2019-nCoV) 3C-like protease (3CL pro) using the crystal structure of the highly similar (96% identity) ortholog from the SARS-CoV. All residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. Comparison of the polyprotein PP1AB sequences showed 86% identity. The 3C-like cleavage sites on the coronaviral polyproteins are highly conserved. Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS-CoV enzyme can be conferred on its SARS-CoV-2 counterpart. With the 3CL pro molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache. The drugs Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes.

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Since the emergence of a novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported from Wuhan, China, neither a specific vaccine nor an antiviral drug against SARS-CoV-2 has become available. However, a combination of two HIV-1 protease inhibitors, lopinavir and ritonavir, has been found to be effective against SARS-CoV, and both drugs could bind well to the SARS-CoV 3C-like protease (SARS-CoV 3CLpro). In this work, molecular complexation between each inhibitor and SARS-CoV-2 3CLpro was studied using all-atom molecular dynamics simulations, free energy calculations, and pair interaction energy analyses based on MM/PB(GB)SA and FMO-MP2/PCM/6-31G* methods. Both anti-HIV drugs interacted well with the residues at the active site of SARS-CoV-2 3CLpro. Ritonavir showed a somewhat higher number atomic contacts, a somewhat higher binding efficiency, and a somewhat higher number of key binding residues compared to lopinavir, which correspond with the slightly lower water accessibility at the 3CLpro active site. In addition, only ritonavir could interact with the oxyanion hole residues N142 and G143 via the formation of two hydrogen bonds. The interactions in terms of electrostatics, dispersion, and charge transfer played an important role in the drug binding. The obtained results demonstrated how repurposed anti-HIV drugs could be used to combat COVID-19.

RESUMO

OBJECTIVE: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS. METHODS: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS. RESULTS: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CLpro), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CLpro inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches. CONCLUSION: RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.

RESUMO

AIMS: The severe acute respiratory syndrome coronavirus 2, better known as COVID-19 has become the current health concern to the entire world. Initially appeared in Wuhan, China around December 2019, it had spread to almost 187 countries due to its high contagious nature. Precautionary measures remain the sole obliging tactic to cease the person to person transmissions till any effective method of treatment or vaccine is developed. Amidst the pandemic, research and development of new molecule is labour-intensive and tedious process. Drug repurposing is the concept of identifying therapeutically potent molecule from the library of pre-existing molecules. MATERIALS AND METHODS: In the present study, 61 molecules that are already being used in clinics or under clinical scrutiny as antiviral agents are surveyed via docking study. Docking study was performed using Maestro interface (Schrödinger Suite, LLC, NY). KEY FINDINGS: Out of these 61 molecules, 37 molecules were found to interact with >2 protein structures of COVID-19. The docking results indicate that amongst the reported molecules, HIV protease inhibitors and RNA-dependent RNA polymerase inhibitors showed promising features of binding to COVID-19 enzyme. Along with these, Methisazone an inhibitor of protein synthesis, CGP42112A an angiotensin AT2 receptor agonist and ABT450 an inhibitor of the non-structural protein 3-4A might become convenient treatment option as well against COVID-19. SIGNIFICANCE: The drug repurposing approach provide an insight about the therapeutics that might be helpful in treating corona virus disease.

RESUMO

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified ß-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.

RESUMO

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that is involved in severe diarrhea disease in piglets, causing considerable agricultural and economic loss in China. The emergence of this new coronavirus increases the importance of understanding SADS-CoV as well as antivirals. Coronaviral proteases, including main proteases and papain-like proteases (PLP), are attractive antiviral targets because of their essential roles in polyprotein processing and thus viral maturation. Here, we describe the biochemical and structural identification of recombinant SADS papain-like protease 2 (PLP2) domain of nsp3. The SADS-CoV PLP2 was shown to cleave nsp1 proteins and also peptides mimicking the nsp2|nsp3 cleavage site and also had deubiquitinating and deISGynating activity by in vitro assays. The crystal structure adopts an architecture resembling that of PLPs from other coronaviruses. We characterize both conserved and unique structural features likely directing the interaction of PLP2 with the substrates, including the tentative mapping of active site and other essential residues. These results provide a foundation for understanding the molecular basis of coronaviral PLPs' catalytic mechanism and for the screening and design of therapeutics to combat infection by SADS coronavirus.

RESUMO

BACKGROUND: Hepatitis C Virus (HCV) infection is a major public health concern across the globe. At present, direct-acting antivirals are the treatment of choice. However, the long-term effect of this therapy has yet to be ascertained. Previously, fluoroquinolones have been reported to inhibit HCV replication by targeting NS3 protein. Therefore, it is logical to hypothesize that the natural analogs of fluoroquinolones will exhibit NS3 inhibitory activity with substantially lesser side effects. METHOD: In this study, we tested the application of a recently devised integrated in-silico Cheminformatics-Molecular Docking approach to identify physicochemically similar natural analogs of fluoroquinolones from the available databases (Ambinter, Analyticon, Indofines, Specs, and TimTec). Molecular docking and ROC curve analyses were performed, using PatchDock and Graphpad software, respectively, to compare and analyze drug-protein interactions between active natural analogs, Fluoroquinolones, and HCV NS3 protein. RESULT: In our analysis, we were able to shortlist 18 active natural analogs, out of 10,399, that shared physicochemical properties with the template drugs (fluoroquinolones). These analogs showed comparable binding efficacy with fluoroquinolones in targeting 32 amino acids in the HCV NS3 active site that are crucial for NS3 activity. Our approach had around 80 % sensitivity and 70 % specificity in identifying physicochemically similar analogs of fluoroquinolones. CONCLUSION: Our current data suggest that our approach can be efficiently applied to identify putative HCV drug inhibitors that can be taken for in vitro testing. This approach can be applied to discover physicochemically similar analogs of virtually any drug, thus providing a speedy and inexpensive approach to complement drug discovery and design, which can tremendously economize on time and money spent on the screening of putative drugs.

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Zika flavivirus is suspected to cause Guillain-Barre syndrome in adults and microcephaly, along with other congenital abnormalities in infants. Presently, no vaccines or therapeutics are available. Here, we report novel compounds identified by high-throughput virtual screening of Maybridge chemical database and molecular docking studies. We selected viral enzyme NS2B/NS3 serine protease as the therapeutic target because of its important role in viral replication. We selected seven potential compounds as antiviral drug candidates because of their high GOLD fitness score, high AutoDock Vina score, or X-Score binding energy and analyzed the strength of molecular interactions between the active site amino acids and selected compounds. Our study also provides a foundation for similar studies for the search of novel therapeutics against Zika virus.

RESUMO

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world's population, especially in the tropics and subtropics, is at risk of infection. Every year, 50-100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 µM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.

RESUMO

Hepatitis C virus (HCV) is a positive-strand RNA virus replicating in a membranous replication organelle composed primarily of double-membrane vesicles (DMVs) having morphological resemblance to autophagosomes. To define the mechanism of DMV formation and the possible link to autophagy, we conducted a yeast two-hybrid screening revealing 32 cellular proteins potentially interacting with HCV proteins. Among these was the Receptor for Activated Protein C Kinase 1 (RACK1), a scaffolding protein involved in many cellular processes, including autophagy. Depletion of RACK1 strongly inhibits HCV RNA replication without affecting HCV internal ribosome entry site (IRES) activity. RACK1 is required for the rewiring of subcellular membranous structures and for the induction of autophagy. RACK1 binds to HCV nonstructural protein 5A (NS5A), which induces DMV formation. NS5A interacts with ATG14L in a RACK1 dependent manner, and with the ATG14L-Beclin1-Vps34-Vps15 complex that is required for autophagosome formation. Both RACK1 and ATG14L are required for HCV DMV formation and viral RNA replication. These results indicate that NS5A participates in the formation of the HCV replication organelle through interactions with RACK1 and ATG14L.

RESUMO

Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

RESUMO

Zika virus is a positive single-strand RNA virus whose replication involved RNA unwinding and synthesis. ZIKV NS3 contains a helicase domain, but its enzymatic activity is not fully characterized. Here, we established a dsRNA unwinding assay based on the FRET effect to study the helicase activity of ZIKV NS3, which provided kinetic information in real time. We found that ZIKV NS3 specifically unwound dsRNA/dsDNA with a 3' overhang in the 3' to 5' direction. The RNA unwinding ability of NS3 significantly decreased when the duplex was longer than 18 base pairs. The helicase activity of NS3 depends on ATP hydrolysis and binding to RNA. Mutations in the ATP binding region or the RNA binding region of NS3 impair its helicase activity, thus blocking viral replication in the cell. Furthermore, we showed that ZIKV NS5 interacted with NS3 and stimulated its helicase activity. Disrupting NS3-NS5 interaction resulted in a defect in viral replication, revealing the tight coupling of RNA unwinding and synthesis. We suggest that NS3 helicase activity is stimulated by NS5; thus, viral replication can be carried out efficiently. Our work provides a molecular mechanism of ZIKV NS3 unwinding and novel insights into ZIKV replication.

RESUMO

BACKGROUND: Zika virus (ZIKV) has emerged as a global pathogen causing significant public health concerns. China has reported several imported cases where ZIKV were carried by travelers who frequently travel between China and ZIKV-endemic regions. To fully characterize the ZIKV strains isolated from the cases reported in China and assess the risk of ZIKV transmission in China, comprehensive phylogenetic and genetic analyses were performed both on all ZIKV sequences of China and on a group of scientifically selected ZIKV sequences reported in some of the top interested destinations for Chinese travelers. METHODS: ZIKV genomic sequences were retrieved from the National Center for Biotechnology Information database through stratified sampling. Recombination event detection, maximum likelihood (ML) phylogenetic analysis, molecular clock analysis, selection pressure analysis, and amino acid substitution analysis were used to reconstruct the epidemiology and molecular transmission of ZIKV. RESULTS: The present study investigated 18 ZIKV sequences from China and 70 sequences from 16 selected countries. Recombination events rarely happens in all ZIKV Asian lineage. ZIKV genomes were generally undergone episodic positive selection (17 sites), and only one site was under pervasive positive selection. All ZIKV imported into China were Asian lineage and were assigned into two clusters: Venezuela-origin (cluster A) and Samoa-origin cluster (cluster B) with common ancestor from French Polynesia. The time of most recent common ancestors of Cluster A dated to approximately 2013/11 (95% highest posterior density [HPD] 2013/06, 2014/03) and cluster B dated to 2014/08 (95% HPD 2014/02, 2015/01). Cluster B is more variable than Cluster A in comparison with other clusters, but no varied site of biological significance was revealed. ZIKV strains in Southeast Asia countries are independent from strains in America epidemics. CONCLUSIONS: The genetic evolution of ZIKV is conservative. There are two independent introductions of ZIKV into China and China is in danger of autochthonous transmission of ZIKV because of high-risk surrounding areas. Southeast Asia areas have high risk of originating the next large-scale epidemic ZIKV strains.

RESUMO

Telaprevir is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease. it is used for the treatment of HCV infection in combination with peginterferon alfa and ribavirin. In the present work, the E-Z isomerization process of telaprevir in solution was revealed by online HPLC-DAD (diode array detector)-MS, variable-temperature and variable-gradient experiments. The molecular geometry information of the two isomers was established by molecular mechanics calculations, and good correlation between the two isomers' UV-vis spectra and their molecular geometry information was also discovered. In addition, it was revealed by molecular docking that the two isomers have different affinities to HCV NS3â¢4A protease, and the Z isomer, the minor form of telaprevir in solution, is the more effective inhibitor of HCV NS3â¢4A protease. The investigation can provide more structure information about telaprevir in solution and in the binding process of HCV NS3â¢4A protease.

RESUMO

Chikungunya virus (CHIKV) is an important arthritogenic human pathogen that is already circulating in both hemispheres. In the present study, we substituted VLoop, located on the surface of nsP2, by other amino acid sequences. These modifications had deleterious effects on viral nuclear functions and made CHIKV incapable of interfering with the induction of type I interferon and the antiviral response in both mouse and human cells. Importantly, the identified mutations have no significant effects on the synthesis of virus-specific RNAs and viral structural proteins. The designed mutants induced a few orders of magnitude lower viremia but remained highly immunogenic in mice. Thus, the proposed modifications of nsP2 can additionally improve the safety of the attenuated strain CHIKV 181/25. Furthermore, defined mutations in the macro domain of another nonstructural protein, nsP3, additionally reduce cytopathogenicity of nsP2 mutants in human cells, and can be potentially applied for CHIKV attenuation.

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